Intronic locus determines SHROOM3 expression and potentiates renal allograft fibrosis
Madhav C. Menon, … , John Cijiang He, Barbara Murphy
Madhav C. Menon, … , John Cijiang He, Barbara Murphy
Published December 1, 2014
Citation Information: J Clin Invest. 2015;125(1):208-221. https://doi.org/10.1172/JCI76902.
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Research Article Nephrology

Intronic locus determines SHROOM3 expression and potentiates renal allograft fibrosis

Abstract

Fibrosis underlies the loss of renal function in patients with chronic kidney disease (CKD) and in kidney transplant recipients with chronic allograft nephropathy (CAN). Here, we studied the effect of an intronic SNP in SHROOM3, which has previously been linked to CKD, on the development of CAN in a prospective cohort of renal allograft recipients. The presence of the rs17319721 allele at the SHROOM3 locus in the donor correlated with increased SHROOM3 expression in the allograft. In vitro, we determined that the sequence containing the risk allele at rs17319721 is a transcription factor 7–like 2–dependent (TCF7L2-dependent) enhancer element that functions to increase SHROOM3 transcription. In renal tubular cells, TGF-β1 administration upregulated SHROOM3 expression in a β-catenin/TCF7L2–mediated manner, while SHROOM3 in turn facilitated canonical TGF-β1 signaling and increased α1 collagen (COL1A1) expression. Inducible and tubular cell–specific knockdown of Shroom3 markedly abrogated interstitial fibrosis in mice with unilateral ureteric obstruction. Moreover, SHROOM3 expression in allografts at 3 months after transplant and the presence of the SHROOM3 risk allele in the donor correlated with increased allograft fibrosis and with reduced estimated glomerular filtration rate at 12 months after transplant. Our findings suggest that rs17319721 functions as a cis-acting expression quantitative trait locus of SHROOM3 that facilitates TGF-β1 signaling and contributes to allograft injury.

Authors

Madhav C. Menon, Peter Y. Chuang, Zhengzhe Li, Chengguo Wei, Weijia Zhang, Yi Luan, Zhengzi Yi, Huabao Xiong, Christopher Woytovich, Ilana Greene, Jessica Overbey, Ivy Rosales, Emilia Bagiella, Rong Chen, Meng Ma, Li Li, Wei Ding, Arjang Djamali, Millagros Saminego, Philip J. O’Connell, Lorenzo Gallon, Robert Colvin, Bernd Schroppel, John Cijiang He, Barbara Murphy

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Figure 5

SHROOM3 facilitates canonical TGF-β1/SMAD3 signaling and profibrotic gene expression in vitro.

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SHROOM3 facilitates canonical TGF-β1/SMAD3 signaling and profibrotic gen...
(A and B) Control- and SHROOM3-transfected PRCECs were treated or not with TGF-β1 (5 ng/ml). (A) Relative COL1A1 transcript levels, by RT-PCR (normalized to GAPDH), after 24 hours of TGF-β1 treatment (n = 4 sets). (B) WBs of phospho- and total SMAD3, SHROOM3 (anti-V5 tag), and β-actin at 30 minutes of TGF-β1 treatment. (C and D) Lentiviral shRNA–mediated knockdown of SHROOM3 was performed in PRCECs. (C) Relative COL1A1 transcript levels, by RT-PCR (normalized to GAPDH), after 24 hours of TGF-β1 treatment. (D) Cells were treated with TGF-β1 for 30 minutes. Shown are WBs of phospho-SMAD3, SMAD3, SHROOM3, and β-actin of a representative experiment of 4. (E–G) PRCECs transfected with SHROOM3 or control were treated with 10 or 20 μM HF for 12 hours in serum-starved medium. (E) Phosphorylation of SMAD3 was assayed in lysates after 30 minutes of TGF-β1 treatment. Representative WB of phospho-SMAD3, SMAD3, SHROOM3, and β-actin is shown. (F) Densitometry of relative phospho-SMAD3 (n = 3 sets). (G) 24 hours after transfection, PRCECs were continuously treated with HF for 24 hours in serum-starved medium. TGF-β1 or vehicle was added for the final 12 hours. Relative COL1A1 mRNA, by RT-PCR (normalized to GAPDH), was determined after 48 hours of transfection (n = 3). (A, C, F, and G) Values are mean ± SEM. *P < 0.05; **P < 0.001; ***P < 0.0001, ANOVA with post-test Bonferroni comparisons.